Various methods have been developed in the prior art for overcoming the diffraction limit in microscopy. From WO 2006/0127692 or DE 102006021317 A1 there is known a method, abbreviated as PALM (photo activated localization microscopy), which uses a marking substance for the imaging of a specimen, which can be activated by means of optical radiation. Only in the activated state can the marking substance emit a particular fluorescent radiation. Nonactivated molecules of the labeling substance, even after exposed to excitation radiation, emit no or substantially no noticeable fluorescent radiation with the defined properties. One therefore generally refers to the activation radiation as a switching signal. In the PALM method, the switching signal is supplied such that at least some of the activated marking molecules are so far away from neighboring activated marking molecules that they are separate in regard to the optical resolution of the microscope or can be separated afterwards by image processing methods. One speaks of fluorescence markers being isolated, and this step is also called an isolation step. It is enough to isolate a subset of the total group of fluorescence markers. The specimen is thus imaged; one obtains a single image of the specimen in which at least some of the fluorescence markers radiate in an isolated manner. Then, for each fluorescence marker, the center of the recorded radiation distribution is determined, which is not pointlike of course, due to the resolution limit. In this way, the position of the fluorescence marker is mathematically localized with higher precision than the optical resolution actually allows. This step is called the localization step.
The steps of isolation and localization are done repeatedly, so that one obtains several individual images. Ideally, each fluorescence marker is isolated once in at least one individual image. The position indications determined from the individual images make it possible to produce an overall image, containing the position indications of the individual fluorescence markers each time with an accuracy beyond the optical resolution. Such an image, having an accuracy beyond the optical resolution, is known as a high-resolution image.
The PALM principle utilizes statistical effects for isolating the fluorescence markers. In the case of a fluorescence marker which can be activated by the switching signal with a given intensity to emit fluorescence, one can ensure, by adjusting the intensity of the switching signal, that the probability of activating fluorescence markers present in a given surface region of the specimen, is so low that there are sufficient subregions in the imaged specimen in which at least some isolated fluorescence markers can be excited to emit fluorescent radiation within the optical resolution. The excitation of the so activated specimen then results in isolated radiating fluorescence markers.
PALM has been modified in regard to the activation, i.e., the supplying of the switching signal. Thus, for example, in the case of molecules having a long-lived nonfluorescing state and a short-lived fluorescing state, a separate activation with activation radiation different in spectrum from the excitation radiation is not even necessary. Instead, the specimen is at first illuminated with excitation radiation of high intensity so that the overwhelming majority of the molecules are brought into the nonfluorescent long-lived state (e.g., a triplet state). Then the remaining fluorescent molecules are isolated, at least in part.
The PALM principle has in the meantime also been referred to by other acronyms in the technical literature, such as STORM, etc. In this specification, we shall use the acronym PALM to identify all microscopy techniques which accomplish a high resolution by first isolating fluorescence markers and then localizing them. The PALM method has the advantage that no high spatial resolution is needed for the excitation. A simple wide-field illumination is possible.
The PALM principle achieves high resolution in 2D or laterally, i.e., transversely to the imaging direction, since the localization can only be done for fluorescence markers which are isolated in projection onto a plane lying perpendicular to the imaging direction. Fluorescence markers lying one behind another along the imaging direction, i.e., in the depth direction, cannot be distinguished with the PALM principle per se. The first experimental realizations of the PALM method therefore used a TIRF illumination, in order to make sure that fluorescence markers are only excited from a sharply defined depth region, which is substantially less than the depth of field of the imaging optics used.
Meanwhile, additional methods and approaches have been developed in the prior art which accomplish a 3D localization microscopy in which fluorescence markers are also isolated and localized in the third spatial dimension, i.e., the depth direction in regard to the imaging. By “depth direction” is meant the direction along the incident light, i.e., along the optical axis.
The publication of B. Huang et al., Science 319, page 810, 2008, describes an imaging beam path for the PALM principle in which a weak cylindrical lens is placed, causing a deliberate astigmatic distortion in the image. In this way, the image of each fluorescence marker is elliptically distorted on the camera whenever the fluorescence markers are located above or below the focal plane, representing a point of symmetry of the point spread function of the specimen imaging. From the orientation and the strength of the distortion one can gain information as to the depth position of the radiating fluorescence marker. One drawback of this method is that the local surroundings and orientation of a molecular dipole can also result in a distortion of the image of the radiating fluorescence marker, which has nothing to do with the depth position. Such radiating fluorescence markers then are given a false depth value, depending on their spatial position.
A different approach is taken by the publication of Shtengel et al., PNAS 106, page 3125, 2009. Here, photons emitted by the radiating fluorescence markers are caused to interfere with each other. For this, two objectives are used, mounted in a 4π configuration, which observe the radiating fluorescence markers at the same time. With the aid of a special 3-way beam splitter, the radiation from the so obtained partial beam paths is brought into interference. Each of the obtained images is detected with a camera, and the intensity relations of the images give an idea as to the depth position.
In the publication of Toprak et al., Nanolet. 7, pages 2043-2045, 2007, and also according to Juette et al., Nature Methods 5, page 527, 2008, a 1:1 beam splitter is installed in the imaging beam path, which splits up the image of the specimen into two subimages, which are detected independently. In addition, an optical path length difference is introduced in one of the partial beam paths after the beam splitter so that the two partial beam paths project two object planes which are separated by around half or the whole optical minimum resolution in the depth direction. The depth position of a fluorescence marker situated between these two object planes is found by analyzing the two partial images of this fluorescence marker (e.g., in terms of the width of the point spread). The method requires two highly resolved partial images and a subpixel-exact superpositioning of these two partial images. A modification of this approach which drastically reduces the adjustment expense is known from DE 102009060490 A1.
Another principle for gaining depth information for 3D localization microscopy is found in DE 102010044031 A1. This uses so-called light sheet illumination for the excitation and/or switching radiation, as described for example in the publication of P. Keller and E. Stelzer, “Quantitative In Vivo Imaging of Entire Embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy”, Current Opinion in Neurobiology, 2008, Vol. 18, pages 624-632. The specimen is illuminated in succession by two axially offset, but overlapping light sheets. Molecules which emit fluorescent radiation in both positions of the light sheets must necessarily lie in the overlap region of the two light sheet positions. A suitable filtering is therefore done. In this way, the depth selection can be substantially boosted beyond the thickness of the light sheet. The thickness of the overlap region is critical to the filtering. The drawback to this approach is that twice the number of individual images have to be recorded for the localization, namely, for each light sheet position the number of single images that would be needed during traditional PALM imaging. The precise adjustment of the offset of the light sheets and especially the reproducibility of the adjustment is also important for the thickness of the overlap region and thus for the depth resolution. Finally, as a rule no meaningful resolution is possible any more within the filtered overlap region. Thus, the overlap region defines a kind of measurement uncertainty in regard to the depth indication. Fluorescence markers lying outside the overlap region cannot be specified in terms of their depth position, so that a detection of a region which is larger than the overlap region ultimately requires a scanning of the specimen.
In PAL microscopy, furthermore, an unwanted irradiation of the fluorescence markers can be a disadvantage, since the fluorescence markers often can only go through a very limited number of activation and/or excitation cycles. In this regard, any irradiation not utilized for a high-resolution imaging is undesirable.
One principle of depth resolution in localization microscopy employs a deliberate distortion of the point spread function (hereafter also abbreviated as PSF) of the imaging. Such an approach is described, for example, in WO 2012/039636, which modified the imaging of the specimen such that an image distortion is produced, which is dependent on the depth position. For example, the ideally spherical point spread function is modified so that for the imaging of a light spot we get two adjacent lobes instead of a diffraction disk, which are offset relative to each other depending on the depth position of the light spot being imaged.
The publication of Pavani et al., PNAS 106, page 2995, 2009, proposes modifying the point spread function by a 3D phase modulator in the imaging to produce a double helix structure. The point images of individual luminescent marking molecules than become double spots, and their depth position is coded in the angle orientation of the shared axis of the double spots.
What is common to the above mentioned 3D localization methods is the problem of a limited capture range, i.e., the range in which molecules can be detected and localized without intervention at the objective. Typically, this is at most 1 to 1.5 μm. This small capture range has the consequence that several slices or sections have to be recorded for a given z-dimension of the specimen being studied. This results in long recording time. Furthermore, generally while producing such slices areas of the specimen are excited which are not even used for the imaging. This represents an unwanted irradiation in the sense described above. Fluorescence emitters which undergo the activation and/or excitation cycles but were not used for the localization generally can no longer be used for another measurement, so that a complete imaging of the specimen may be difficult if not impossible.